Screening molecules with anti-prion activity: kits, methods and screened molecules

ABSTRACT

The invention concerns screening molecules with anti-prion activity. More particularly, it concerns kits for screening molecules with anti-prion activity characterized in that they comprise in combination a [PSI+], phenotype yeast, an antibiogram and an agent for purifying prions at sub-efficient doses, said yeast including the adel-14 allele of the ADE1 gene and an inactivated ERG6 gene, the screening methods, and a family of molecules with anti-prion activity isolated by the inventive screen. The invention is applicable to anti-prion agents for producing medicines in particular for treating neurodegenerative diseases involving protein aggregates.

This application is a U.S. National Stage of International ApplicationNo. PCT/FR2003/003101, filed Oct. 20, 2003, and published on Apr. 29,2004, as WO 2004/035813. This application claims priority to FrenchPatent Applications Nos. FR02/13022, filed Oct. 18, 2002, andFR03/08289, filed Jul. 7, 2003, hereby incorporated by reference for allpurposes.

The present invention relates to screening of molecules with anti-prionactivity. It relates more particularly to kits for screening moleculeswith anti-prion activity, methods of screening, and a family ofmolecules with anti-prion activity revealed using the screen accordingto the invention.

Prions are infectious proteins responsible for certainneuro-degenerative diseases of spongiform encephalopathy type inmammals, such as Creutzfeldt-Jakob's disease in humans or also theso-called “mad cow disease” in bovines or “scrapie” in ovines. Thesedifferent diseases are caused by unconventional infectious agents:unlike traditional infectious agents (bacteria, viruses for example),they contain no nucleic acids. Professor Stanley Prusiner formulated the“protein-only” hypothesis, according to which the infectious agent wouldbe constituted only by a protein. This protein exists naturally in cellsin a normal (or PrP^(c)) form, i.e. soluble, essentially in the form ofan α helix and non-aggregated, therefore functional. Under certain stillunknown conditions, this protein can be converted to a prion (orPrP^(sc)) form. In this prion form, the protein forms insolubleaggregates, essentially in the form of β sheets. The infectiouscharacter of this PrP^(sc) prion conformation would result from the factthat, apart from the characteristics indicated previously, the proteinin prion form also gains the ability to catalyze the passage from thenormal Prp^(c) cell form to the PrP^(sc) prion form in a “snowball”-typemechanism. Baker's yeast Saccharomyces cerevisiae contains severalproteins that behave like prions (Fernandez-Bellot and Cullin, 2001).Since as long ago as the 1960s, two unconventional genetic mechanismshave been described. In 1994, the corresponding [PSI+] and [URE3]phenotypes were proposed as resulting from the autocatalyticinactivation of the Sup35p and URE2p proteins respectively. These prionproteins therefore have a priori a mechanistic analogy with mammalsystems deleterious to public health. Like the PrP protein, the “normal”Sup35p protein passes from a soluble state to an insoluble andaggregated state as soon as the protein is in contact with anotherSup35p protein in prion form. This aggregated state is verified both bycentrifugation experiments and by intracellular localizationexperiments. Yeast prions can be eliminated (“cured”) by a strong dose(1 to 5 mM) of guanidium chloride. As a result of such a treatment(which must applied to at least six to ten generations), the proteinaggregates generated by the presence of the prions disappear and theprotein in question (Sup35p, for example) is found in a normal, soluble,functional form but having retained the capability of being converted toa prion form should it again come into contact with another Sup35pprotein in such a state.

The Sup35p protein, in a heterodimeric complex with the Sup45p protein,forms a translation termination factor. This factor recognizes the opalstop codons (UGA). In its normal cell form (soluble and active) in the[psi−] strains, Sup35p, in combination with Sup45p effectivelyterminates translation at the level of these opal codons. In a [PSI+]strain where the Sup35p protein is in prion form, it is mostly presentin the form of insoluble aggregates. Being unable to bind to Sup45p, itis thus non-functional in the translation termination. A small fractionof all of the cellular Sup35p proteins however remains soluble in these[PSI+] cells where it makes it possible, in a complex with Sup45p, toensure a “minimum translation termination service”, a service essentialto the survival of the yeast. A colorimetric system making it possibleto detect, in an indirect fashion, the form in which the Sup35p proteinis present: normal or prion, has been produced from these findings. Thissystem, which has been described for a long time (see the article onsynthesis by Fernandez-Bellot and Cullin, 2001), is based on the use ofthe adel-14 allele of the ADE1 gene, coding for an enzyme of the adeninebiosynthesis route: SAICAR synthetase. This enzyme catalyzes theformation of 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide(SAICAR) from 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Theadel-14 allele contains an opal codon in the reading frame of the ADE1gene. In a [psi−] strain, Sup35p in combination with Sup45p willtherefore stop the translation of the ADE1 gene at the level of thisstop codon. The protein adel-14p thus translated will be truncated andtherefore non-functional. As a result the substrates upstream of theAde1p enzyme will accumulate, in particular the 5-aminoimidazoleribonucleotide (AIR) The AIR being oxidized to a red-coloured compound,the colonies formed by the [psi−]. cells will be red in colour.Moreover, these cells will be auxotrophic for adenine. Conversely, in a[PSI+] strain, the protein Sup35p is essentially present in the form ofaggregates therefore incapable of being combined with Sup45p in order tostop translation at the level of the opal codon of the adel-14 allele ofthe ADE1 gene. As a result, the ribosomes will pause at the level ofthis stop codon before resuming their translation activity(readthrough). A certain quantity of functional Ade1p protein willtherefore be synthesized, the cells will be autotrophic for adenine andwill form white to pink-coloured colonies.

In an article which appeared in P.N.A.S, Prof. Stanley Prusiner's teamdiscloses a test for detecting molecules with anti-prion activity (Korthet al., 2001). This test is carried out on a mammal model (murineneuroblastomas infected with PrP^(sc)) The safety conditions (P3laboratory) and cell culture conditions (significant handling) do notallow high-throughput screening to be carried out.

The Application WO 98/30909 also describes a process for screeningmolecules with anti-prion activity carried out on rodents infected withan unconventional transmissible agent. This screening method has thesame limits as the method described in P.N.A.S.

The inventors' work has led them to produce a high-throughput screeningsystem in order to detect molecules possessing an anti-prion activity,based on the calorimetric reporter system of the protein Sup35p,described above.

The present invention therefore relates to a kit for screening moleculeswith an anti-prion activity, characterized in that it comprises incombination a yeast of phenotype [PSI+], an antibiogram and a prioncuring agent in sub-effective doses, said yeast having the adel-14allele of the ADE1 gene as well as an inactivated ERG6 gene.

Although based on yeast prions, the kit according to the invention makesit possible to isolate molecules active against mammal prions. Example 7below shows that the most active molecules isolated by Prof. Prusineralso have an activity in the screen according to the invention.

However, numerous differences are observed between yeast prions andmammal prions. In an article in the journal “Cellular and Molecular LifeSciences”, Professor C. Cullin proposes, even in view of thesedifferences, distinguishing yeast prions from mammal prions by using theterm “propagons”. As particular differences between “prions” (mammal)and “propagons” (yeast), there can be mentioned the cytoplasmiccharacter of propagons whereas the mammal PrP prion is a proteinanchored to the plasmic membrane, the pathological character of mammalprions, as well as a certain number of biophysical differences (ternaryand quaternary structure, reversibility of the curing etc.)

One of the main advantages of such a screen resides in its completeharmlessness which allows it to be carried out in a standard level L2molecular biology laboratory, and not, as required in the previoustechniques, in a level P3 laboratory.

Moreover, the great ease of use and very low cost of this kit make itpossible carry out high-throughput screening. The use of antibiogrampellets, which allow the diffusion of the product by creating aconcentration gradient, moreover makes it possible to test amultiplicity of concentrations in a single experiment, unlike thestandard tests, in which only one concentration is tested. For eachmolecule the anti-prion activity of which is tested, the use of theantibiogram also makes it possible to acquire information on thetoxicity of the product as well as on the activity/concentration ratio,and thus to determine the best effective concentration.

The [PSI+] strain used in the kit according to the invention carries aninactivation of the ERG6 gene. In fact, yeasts are naturally fairlyimpermeable. In particular, the preferred yeast for implementing theinvention, Saccharomyces cerevisiae, has an impermeability such that thecarrying out of a screening process proves particularly ineffectivewithout this inactivation.

The screen analysis method according to the invention is visual thanksto the use of the adel-14 allele. According to the anti-prion activityof the molecule tested, the colonies of cells will have a red, pink orwhite staining. The choice of the strain of yeast can make it possibleto improve the contrast between the colonies. In fact, certain so-called“Strong” strains facilitate visual analysis of the screen. Such strainspossess a strong level of aggregation of the prion forms. In theopposite case, the strain is referred to as “Weak”. The strainspreferred for implementation of the invention are therefore the“Strong”-type strains.

Other yeasts can also be used. As examples there can be mentioned:Kluyveromyces lactis, Pichia methanolica, Saccharomyces ludwigii,Kluyveromyces marxianus, Pichia pastoris, Zygosaccharomyces rouxi,Schizosaccharomyces pombe.

Given the synthetic lethality observed between the inactivation of theERG6 gene and the inactivation of the TRP1 gene, the ERG6 gene can bedeleted using the TRP1 gene as deletion marker.

Advantageously, the kit moreover comprises a prion curing agent atsub-effective doses.

By curing, is meant an elimination of the prion forms from the yeastcells. This elimination can be temporary or permanent.

By way of example, a prion curing agent can be hydrogen peroxide orpreferentially, guanidium chloride.

By sub-effective doses, is meant doses which used alone would notsuffice to eliminate the prions from the yeasts. The values of suchdoses are given, in the examples which follow, for guanidium chloride.

The benefits of the presence of a curing agent at sub-effective dosesare to reinforce the sensitivity of the screen and obtain a bettercontrast.

The kit according to the invention can be used in a method for screeningmolecules with anti-prion activity. This screening method, to which theinvention also relates, is characterized in that it uses a [PSI+]phenotype yeast having the adel-14 allele of the ADE1 gene as well as aninactivated ERG6 gene and comprises the following stages:

-   a. production of a lawn of cells in vitro on a medium complemented    with a sub-effective dose of a prion curing agent,-   b. deposition of the compounds to be tested according to the    antibiogram method,-   c. incubation for approximately 2-4 days at approximately 20-25° C.,    and,-   d. analysis of the staining of the cell colonies.

This method possesses advantages analogous to those of the kit accordingto the invention. It is a visual test, very easy to analyze. Itsimplementation is very simple and inexpensive. The precautions relativeto safety are those of a standard molecular biology laboratory. Itallows mass screening: a single person can manually screen more than 400products per day. Very high-throughput screening would be possible byautomation of the method. The screen result is developed after 7 days,without it being necessary to resort to a lot of handling between day Dand day D+7 (optionally a change in temperature of the incubator).Finally, this method is particularly economical.

One of the yeasts preferred for the implementation of this method isSaccharomyces cerevisiae.

Advantageously, the curing agent of Stage a. is guanidium chloride.

The method can also comprise the following stages:

-   e. incubation for approximately 2-4 days at approximately 2-6° C.,    and/or,-   f. carrying out a secondary screening test.

The incubation at 2-6° C. makes it possible to accentuate the contrastin staining of the colonies.

Preferentially, the secondary screening test can comprise the followingstages:

-   -   construction of a strain of yeast in which the ADE2 gene is        under the control of the DAL5 gene promoter    -   carrying out Stages a. to e. of the methods described above.

Such a secondary screening makes it possible to test very rapidlywhether the molecules isolated during the primary screening can have ageneral effect on the prions in the yeast. In fact, the SUP35 genes(responsible for the [PSI+] prion) and URE2 (responsible for the [URE3]prion) code for enzymes having totally different functions and theprimary sequences of which are very remote.

The invention also covers the molecules isolated by the screening methodaccording to the invention.

In particular, the screening method has made it possible to isolateanti-prion agents having the following formula (I):

-   in which R is an H, NH₂, NHR² group, where R² is an alkyl or    alkylaminoalkyl chain with 1 to 10 carbon atoms, branched or    unbranched,    -   X represents F, Cl, Br, I, CF₃, SR³, OR³, OH, NO₂, COR³, CONH₂,        COOH, COOR³, where R³ is an alkyl group with 1 to 4 carbon        atoms, preferably CH₃.    -   p and n, identical or different, are equal to 0, 1 or 2,    -   q is equal to 0 or 1.

The invention relates in particular to the anti-prion agents of formula(III):

-   in which R′ represents an H, NH₂, NH—(CH₂)₃—N(CH₃)₂,    NH—CH(CH₃)—(CH₂)₃—N(CH₂—CH₃)₂ group,    -   X represents F, Cl, CF₃,    -   p and n, identical or different, are equal to 0, 1 or 2.

This family of molecules, called “Kastellpaolitines” by the inventors,possesses the sought anti-prion activity to a greater or lesser degree.In particular, the chlorinated derivatives of this family areparticularly effective. The best effectivenesses are obtained whenchlorine is placed in position 2, 3 or 4, preferably in position 4 (seeKP1 in the examples which follow).

The invention relates more particularly to the compounds of formula(II):

-   in which R′ represents an H, NH₂, NH—(CH₂)₃—N(CH₃)₂,    NH—CH(CH₃)—(CH₂)₃—N(CH₂—CH₃)₂ group,    -   X represents F, Cl, CF₃,    -   p and n, identical or different, are equal to 0, 1 or 2.        for use as a medicament, and in particular, as an anti-prion        agent.

It also relates to the pharmaceutical compositions comprising atherapeutically effective quantity of at least one compound of formula(II) in which:

-   -   R′ represents an H, NH₂, NH—(CH₂)₃—N(CH₃)₂,        NH—CH(CH₃)—(CH₂)₃—N(CH₂—CH₃)₂ group,    -   X represents F, Cl, CF₃,    -   p and n, identical or different, are equal to 0, 1 or 2.        in combination with at least one pharmaceutically acceptable        vehicle.

Certain compounds of this family are particularly active. These arephenanthridine and 6-aminophenanthridine, as well as their chlorinatedderivatives, in particular when the chlorine is placed in position 8, 9or 10, preferably in position 10 (see in the examples which follow).

Preferentially, in formulae (II) and (III), R′ represents NH₂. In fact,a very good activity of the molecules has been noted when R′ representsNH₂.

The invention also proposes a method for treating neurodegenerativediseases involving protein aggregates, comprising a stage ofadministering to an animal or to a patient a therapeutically effectivequantity of at least one of the compounds of formula (I), (II) or (III)according to the invention.

The anti-prion agents according to the invention are particularly usefulfor obtaining a medicament intended to prevent and/or to treatneurodegenerative diseases, in particular of the protein-aggregationtype, such as the spongiform encephalopathies, Alzheimer's (tau),Parkinson's (α-synuclein) and Huntington's (huntingtin) disease etc.These medicaments can be intended for human or veterinary use, inparticular for domestic (cows, sheep etc.) or wild animals (lynx, theCervidae such as deer, moose etc.).

Other characteristics and advantages of the invention will becomeapparent in the examples below and by referring to the followingfigures:

FIG. 1 relates to the feasibility of the screen,

FIG. 2 illustrates the screening protocol,

FIG. 3 relates to the isolation of the Kastellpaolitines, phenanthridineand to their structure/activity relationship,

FIG. 4 relates to the determination of the activity of thephenanthridine derivatives,

FIG. 5 shows the results of the liquid curing tests,

FIG. 6 relates to the secondary screen based on the [URE3] prion,

FIG. 7 demonstrates the validation of the test with chlorpromazine,quinacrine and verapamil,

FIG. 8 shows the results of the effect of KP1 on the mammal prion in anin vitro model, and,

FIG. 9 relates to a structure/activity study carried out on the moleculeof general formula (II).

EXAMPLE 1 Carrying Out the Screen 1. Material and Methods

Organisms (Saccharomyces cerevisiae) and Culture Media

The [PSI+] haploid yeast strain 74-D694 (Mat a, adel-14, trpl-289,his3-Δ200, ura3-52, leu2-3,112) was used in the development of thescreening method. The strain used is called “Strong” as it has awell-marked phenotype when the translation termination factor Sup35p isin prion or aggregated form.

In order to increase the penetration of the inhibitors, the inventorsgenetically modified this strain by introducing into it a mutation ofthe ERG6 gene. This gene is involved in the biosynthesis of ergosterol,a component of the cell wall of the yeasts. The mutation was produced byinsertion at the level of the chromosome site of the ERG6 gene of a“deletion cassette” corresponding to the TRP1 marker gene flanked by DNAsequences situated upstream and downstream of the coding frame of theERG6 gene. This cassette was produced by PCR using the plasmidpFA6a-kanMX6 as matrix and the oligonucleotides oBM1060 (5′) et oBM1061(3′) as primers. The “Strong” 74-D694 yeast cells having integrated thedeletion cassette (strain called STRg6, deposited at the CNCM on 10thOct. 2002 under number 1-2943) are those which develop on minimum mediadevoid of tryptophan. The mutation Δerg6::TRP1 was then verified by PCRusing the genomic DNA of the strain STRg6 as matrix and theoligonucleotides oBM1030 (5′) and oBM1063 (3′) as primers.

The PCR primers used have the following nucleotide sequences:

(SEQ ID No. 1) oBM1060 5′ CGATTTAAGTTTTACATAATTTAAAAAAACAAGAATAAAATAATAATATAGTAGGCAGCATAAGCGGATCCCCGGGTTAATTAA 3′ (SEQ ID No. 2)oBM1061 5′ CTGCATATATAGGAAAATAGGTATATATCGTGCGCTTTATTTGAATCTTATTGATCTAGTGAATGAATTCGAGCTCGTTTAAAC 3′ oBM1030 5′GGTACCTCGTTCCCGTAC 3′ (SEQ ID No. 3) oBM1063 5′ CAGTCAGAAATCGAGTTCCA 3′(SEQ ID No. 4)

Unless otherwise indicated, the yeast strains are cultured at 30° C. inrich medium (YPDψ) or in minimum medium. Unless explicitly specified,the percentages correspond to a mass/volume ratio. The gelosed form isobtained by the addition of 2% agar.

YPDψ: 1% yeast extract (FISHER®), 2% peptone (GiBCO®) and 2% glucose;

Minimum medium: 0.175% yeast nitrogen base without amino acid andammonium sulphate (DiFCO®), 0.75% ammonium sulphate and 2% glucose. Thismedium is adjusted to pH 6. In order to compensate for possibleauxotrophies, this medium can be completed, after sterilization, by theaddition of amino acids (0.002% L-histidine and/or 0.004% L-leucineand/or 0.003% L-tryptophan) or nitrogenous bases (0.0025% uracil and/or0.008% adenine).

Method for screening substances with anti-prion activity (“Prion HaloAssay”)

The screening method developed is based on the antibiogram principle. Infact, the compounds to be tested are applied to a sterile filter-paperdisc, itself applied to a dish of solid YPDψ medium containing 0.2 mM ofguanidium chloride previously seeded with approximately 5·10⁶ cells ofthe STRg6 strain in order to produce a yeast lawn. This quantity ofseeded cells (from 10⁶ to 10⁷) was optimized in order for each cell tobe able to divide at least 6 times (number of generations necessary tohave an effective curing effect with 3 mM of GuHCl). The addition of asmall quantity of guanidium chloride (0.2 mM), a sub-effective dose foreliminating prions from yeast (the effective dose being of the order of1 to 5 mM) makes it possible to increase the sensitivity of the test(see Results section). The 12 cm square dishes are then incubated for 3days at 23.5° C. in order to allow the appearance and growth of theyeast colonies. These dishes are then stored for 3 days at 4° C. inorder to accentuate the red staining present around the discs soakedwith ingredients active on the prion form of the protein Sup35p.Comparison with the negative controls (application of the solvent of theinhibitors tested) and positive controls (application of a 300 mMguanidium chloride solution, causing effective elimination of the Sup35pproteins in prion form) makes it possible to judge the effectiveness ofa compound. FIG. 2 illustrates the protocol of the screening method: (1)Culture of the STRg6strain; (2) Application and plating with sterileglass beads 3 & 4 mm in diameter, of approximately 106 cells inexponential growth phase on a dish of solid YPDψ medium containing 0.2mM of guanidium chloride: constitution of the cell “lawn”; (3)Application of the sterile filter-paper discs according to a gridallowing the analysis of 32 compounds (including controls) and depositof 20 μl maximum of each of the products to be tested; (4) Incubation;(5) Scanning of the result obtained; (6) Example showing the isolationof a compound having a strong anti-prion activity.

Synthesis of ll-aminodibenzo[b,f][1,4]thiazepines and6-aminophenanthridine

ll-aminodibenzo[b,f][1,4]thiazepines, also called Kastellpaolitines, canbe prepared in a single stage. The synthesis of these products hasalready been described in the publication by Mettey et al., 1997.

2. Results

Principle and Feasibility of the Screen

Guanidium chloride, the only product known to effectively eliminateprions from the yeast Saccharomyces cerevisiae, served not only as apositive control throughout screening, but also for studying thefeasibility of the method as well as developing it. Guanidium chlorideeffectively eliminates the different yeast prions at a dose comprisedbetween 1 and 5 mM (Fernandez-Bellot and Cullin, 2001). Under theseconditions, the curing requires a constant presence of this product forsix to ten generations in exponential growth phase compromising thefeasibility of the screen on a dish such as the inventors wished toachieve.

FIG. 1 shows the feasibility of the screen.

The three left-hand panels: a [PSI+] strain is cultured for 48 hours inthe presence of 5 mM guanidium chloride (with 0.2% DMSO final) or, as acontrol, with only 0.2% DMSO final. At T=0, then every 24 hours, a 10 μldrop (approximately 10⁴ cells) is applied to a dish of rich medium. Theguanidium chloride curing begins to have an effect after 24 hours oftreatment, i.e. after approximately 6 generations (a pink stainingbegins to appear). After 48 hours, i.e. after approximately 12generations, the drop of cells has a clearly red staining, a sign of acomplete curing of the [PSI+] cells.

The middle panel: a few cells are taken at T=48 hours and scratched ontoa fresh medium. Almost all of them form red colonies in the case ofcuring with guanidium chloride.

The right-hand panel: these same cells are pelleted at the bottom of anEppendorf tube after liquid culture. In the case of curing withguanidium chloride, they form a red pellet.

The first stage therefore consisted of determining whether guanidiumchloride could have an effect which can be visualized on a dish of[PSI+] cells with the antibiogram pellet system. Once this stage wascarried out, the inventors developed the optimum temperature, medium anddensity conditions as well as cell type to use (FIG. 2). The strainhaving the best sensitivity is the STRg6 strain cultured at 23.5° C. andin the presence of 200 μM of guanidium chloride. In fact, theintroduction of a sub-effective dose of guanidium chloride into themedium makes it possible to increase the sensitivity of the test.

Screening of a Combinatorial Library

Compounds (approximately 1000) were passed through the screen using theconditions optimized by the inventors (FIG. 2). On each dish, 15 μl ofDMSO are deposited on the filter at the top left (negative control) and15 μl of a 300 mM solution of guanidium chloride in DMSO (positivecontrol) were applied to the filter at the bottom right. The same volume(15 μl) of each of the products of the library (all in 10 mM solution inDMSO) was applied to the remaining filters (thirty for each large squarePetri dish). A positive signal (visualization of a red halo around thesterile filter-paper disc to which the product is applied) was obtainedfor five products. These products correspond to four molecules of thesame family, called “Kastellpaolitines” by the inventors, and to awell-known fifth molecule: phenanthridine.

EXAMPLE 2 Identification of the Kastellpaolitines and Phenanthridine

The chemical structures of the Kastellpaolitines and phenanthridine areshown in FIG. 3B. The panel 3A shows a comparative analysis of the sizeof the red halos obtained with all of these molecules respectively (allapplied in an equivalent quantity: 15 μl of a 10 mM solution in DMSO).This experiment makes it possible to compare the relative activity ofeach of these products. The most active is Kastellpaolitine 1 (or KP1)followed by phenanthridine.

6-aminophenanthridine Synthesis and Test

Comparative analysis of phenanthridine on the one hand, and of theKastellpaolitines on the other hand show several common points betweenthese two groups of molecules (FIG. 3). The different molecules areclassified there from the least active to the most active and theirrespective formulae indicated. All are tri-cyclic, the central ringcontaining in all cases a nitrogen atom with a double bond to anadjacent carbon atom. In contrast, in all the Kastellpaolitines, thecarbon of the central ring which has a double bond to this nitrogen atomcarries an amino group, which is not the case for phenanthridine. Thisobservation led the inventors to want to test 6-aminophenanthridine.

6-aminophenanthridine can be prepared according to the proceduredeveloped by Kessar et al., 1969.

6-aminophenanthridine was therefore passed through the screen accordingto the invention, in comparison with the Kastellpaolitines 1 (KP1) and 5(KP5) as well as phenanthridine. The result is very clear:6-aminophenanthridine is still more active than the Kastellpaolitinesand phenanthridine.

FIG. 4 illustrates the results of this comparison: the activity of6-aminophenanthridine was determined on a dish and compared to that ofphenanthridine. For all the molecules, the same quantity is applied (10μl of a 10 mM solution). In the case of the positive control (guanidiumchloride), the solution used was 300 mM.

As a result, by grafting this amino group, characteristic of theKastellpaolitines onto phenanthridine, the inventors significantlyincreased the activity of the latter.

By following the same approach, the inventors then added a chlorine inposition 8 in 6-aminophenanthridine (6AP) in order to produce6-amino-8-chlorophenanthridine (6A-8CP). This modification againincreased the activity of the compound. Finally, the chlorine inposition 8 was replaced by a trifluoromethyl group in order to produce6-amino-8-trifluoromethylphenanthridine (6A-8tFP). As shown by FIG. 4,the latter modification led to an additional increase in activity and6A-8tFP in fact represents one of the most active compounds.

EXAMPLE 3 Synergy Between Products Isolated Using the Screen andGuanidium Chloride

All the active molecules were isolated in a medium containing a weakdose of guanidium chloride (200 μM/effective dose=4 mM). Taking thiscourse, established during the development of the screen corresponded tothe wish to increase the sensitivity (and therefore the detectionthreshold of the method). The effect of the molecules in mediacontaining more (500 μM) guanidium chloride or not containing any, wasobserved subsequently. Phenanthridine is always active on a mediumwithout guanidium chloride, but its activity increases significantly asa function of the quantity of guanidium chloride (however in a clearlysub-effective dose) in the medium. This result indicates a synergy ofaction between guanidium chloride and phenanthridine. The same resultwas obtained for all the other molecules isolated by the inventors (theKastellpaolitines, 6-aminophenanthridine and its derivatives).

EXAMPLE 4 Verification of Liquid Medium Curing

The inventors then wanted to determine whether the red halos observed inthe yeast test corresponded to [PSI+] prion curing and not to anartefact (for example these red halos could be due to a directinhibition of the biosynthesis chain of adenine by these molecules,which would lead to a accumulation of the AIR). If these moleculeseffectively eliminate the [PSI+] prion, a treatment of [PSI+] cells inliquid culture followed by washing of said cells must allow them to formred colonies on a gelosed medium no longer containing the molecules.These tests were carried out with 6-aminophenanthridine on the wild-type“strong” strain 74-D694.

The liquid medium curing conditions are the following: a [PSI+] strainis cultured for 5 days in liquid medium in the presence of the indicatedquantities of the different products (see FIG. 5). Every 24 hours, analiquot fraction is washed in medium uncontaminated by any product andapplied to a solid gelosed medium (itself also uncontaminated by anyproduct) which is then treated as indicated in FIG. 2.

As shown in FIG. 5, 6-aminophenanthridine is capable of partially curingthe [PSI+] prion from a significant number of cells. The curingeffectiveness can in particular be increased by adding a sub-effectivedose (100 μM) of guanidium chloride to the culture medium. In such aliquid curing, the same synergic effect as that observed in the dishtest is also found.

EXAMPLE 5 Development and Use of a Secondary Calorimetric Screen Basedon the Use of [URE3], Another Yeast Prion

Another rapid dish test was carried out, based on another yeast prion:[URE3]. This test constituted a secondary screen which makes it possibleto generalize the effect of the products isolated during the primaryscreen of another yeast prion. In this way, it is possible to remove themolecules active only against the [PSI+] prion and therefore lessuseful, having a non-general effect.

For the [URE3] prion the haploid strain used is CC34 (Mat a, trpl-1,ade2-1, leu2-3,112, his3-11, 15, ura2:: HIS3).

The NT34 strain which served for the secondary screen was constructedfrom CC34, a strain in which the coding frame of the DAL5 gene has beenreplaced by that of the ADE2 gene using the same method as that used forthe construction of the STRg6 strain. For this purpose a deletioncassette corresponding to the ADE2 gene flanked by DNA sequencessituated upstream and downstream of the coding frame of the DAL5 genewas produced by PCR using genomic DNA of the BY4742 haploid strain (Matα, his3Δ1, leu2ΔO, lys2Δ0, ura3Δ0) as matrix and the oligonucleotides:ACAACAAAACAAGGATAATCAAATAGTGTAAAAAAAAAAATTCAAGATGGATTCTAG AACAGTTGG (SEQID No. 5) (5′), andTATATTCTTCTCTGATAACAATAATGTCAGTGTATCTCACCACTATTATTACTTGTT TTCTAGATAAGC(SEQ ID No. 6) (3′) as primers.

The mutation DAL5::ADE2 was then verified by PCR using the genomic DNAof the NT34 strain as matrix and the oligonucleotides:

ATAGTCTCTGCTCATAG (SEQ ID No. 7) (5′), and GCTTACAGAAATTCTAC (SEQ ID No.8) (3′) as primers.

The NT34 strain (Mat a, trpl-1, ade2-1, leu2-3,112, his3-11,15,ura2::HIS3, DAL5::ADE2) was deposited at the CNCM on 10th Oct. 2002under number 1-2942.

This screen is based on the same colorimetric system as the primaryscreen. In the NT34 yeast strain, the ADE2 gene is no longer under thecontrol of its own promoter, but under that of the DAL5 gene. When theprotein Ure2p is in prion form ([URE3]), the transcription from thepromoter of the DAL5 gene is activated, therefore the ADE2 gene isexpressed, therefore the strains are white and autotrophic for adenine.When the URE2p protein is in the normal form ([URE3-0]), thetranscription from the promoter of the DAL5 gene is repressed, thereforethe ADE2 gene is not expressed, therefore the strains are red andauxotrophic for adenine. When the NT34 strain is treated with 5 mM ofguanidium chloride for approximately ten generations, it forms redcolonies (as expected and as the [PSI+] strain used for the primaryscreening would do). As can be observed in FIG. 6, phenanthridine and6-aminophenanthridine cause the appearance of a red halo when they areapplied to the small filter itself applied to the lawn of cellspreviously plated on the gelosed nutritive medium (same process as forthe primary screen, see FIG. 2). This result suggests that theseproducts are also active on the [URE3] prion. It is to be noted,however, that this secondary screen is clearly less sensitive than theprimary screen. It is therefore very useful for rapidly observingwhether the effect of the molecules isolated during the first screen canbe generalized to other yeast prions but in no event could it besubstituted for the primary screen.

In order to increase cell permeability, the coding sequence of the ERG6gene was also replaced by that of the TRP1 gene. In this strain (SB34),the transcription of ADE2 therefore depends on the state of Ure2p: ifUre2p is inactivated by a prion mechanism ([ure3] cells), the ADE2 geneis actively transcribed whereas in the [ure 3-0] cells, it is not.Therefore, the [URE3] cells of the SB34 strain will form white colonieswhereas the [ure3-0] cells will form red colonies. Because this strainalways contains the ade2-1 allele, it was envisaged that this straincould be [PSI+], such that the red staining could be due to the curingof [PSI+] rather than of [URE3]. This possibility has been excluded byverifying using cytoduction and conjugation that the strain is [URE3].Moreover, the entire coding sequence of the ade2-1 gene was deleted inorder to produce the NT35 strain. This strain also formed whitecolonies, demonstrating again that it is [URE3].

The SB34 strain was constructed by replacing the ERG6 gene in CC34 byPCR amplification of the TRP1 marker and by replacing the coding regionof the DAL5 gene by the ADE2 gene using a method based on PCR bydeletion of the ERG6 gene with the primers(5′-ACAACAAAACAAGGATAATCAAATAGTGTAAAAAAAAAAATTCAAGATGGATTCTAGAACAGTTGG-3′) (SEQ ID No. 9) and 342(5′-TATATTCTTCTCTGATAACAATAATGTCAGTGTATCTCACCACTATTATTACTTGTTTCTAGATAAGC-3′) (SEQ ID No. 10). This gene replacementwas then confirmed by growth on the SD-Ade medium, in the absence ofgrowth on the USA medium (as provided for a dal5Δ strain) and byanalytic PCR on the genomic DNA. The [URE3] phenotype of this strain wasverified by cytoduction: among 30 cytoduction agents, 26 were capable ofgrowing on USA medium, showing that they were [URE3]. The NT35 strainwas constructed by replacing the ade2-1 gene in the SB34 strain by themarker KanMX amplified by PCR and by verifying the successfulreplacement of the gene by analytic PCR on the genomic DNA.

EXAMPLE 6 Verification of Liquid Medium [URE3] Curing

Two types of experiments were carried out in order to verify that theeffect observed on dishes with the NT34 strain corresponds to curing.Firstly, cells in the zones surrounding the filter were recovered forthe negative (DMSO), and positive (guanidium chloride) control forphenanthridine and for 6-aminophenanthridine. These cells were thenscratched onto a fresh medium free of all these molecules. The cellsrecovered around the filters all form red colonies, with the exceptionof those collected around the negative control. This result shows thatthe red staining observed on dishes for the NT34 strain corresponds tocuring and not to an artefact linked to inhibition of an enzyme of thebiosynthesis route of adenine (in this case, the red staining would belost on a medium without inhibitor). The curing effect of phenanthridineand 6-aminophenanthridine was also directly verified on the [URE3]prion. [URE3] cells of the CC34 strain grow on a medium called USAwhereas cured ([ure3-0]) cells are incapable of growing on this medium.The inventors examined the ability of [URE3] cells treated with 200 μMof guanidium chloride (negative control), 5 μM of guanidium chloride(positive control) or with different doses of 6-aminophenanthridine(alone or in combination with 200 μM of guanidium chloride) to grow on aUSA medium. 6-aminophenanthridine is capable of curing the [URE3] prionin a significant manner and, just as for the [PSI+] prion, this effectis accentuated by a low dose of guanidium chloride (200 μM). Theseresults, apart from the fact that they validate the secondary screenwith the NT34 strain, suggest that the effect of the inhibitors revealedby said screen should be general on all yeast prions.

EXAMPLE 7 Validation of the Screen with Two Molecules Active on theMammal Prion PrP: Chlorpromazine and Guinacrine

The laboratory of Stanley Prusiner, who first put forward the“protein-only” hypothesis and was awarded the Nobel prize in 1997, hasisolated a certain number of molecules active on the mammal prion PrPusing a system of murine cells (neuroblastomas) chronically infectedwith the prion PrP^(sc) (Korth et al., 2001). This system, due to itslabour-intensiveness and its complexity, does not allow mass screeninglike that developed by the inventors. Thus the approach of StanleyPrusiner's group was to test one-by-one, from the molecules already usedas medicaments, those which pass the blood-brain barrier. Certainmolecules, such as in particular quinacrine (used as an anti-malarialdrug for a long time) or chlorpromazine (an antidepressant) have aparticular activity in their system. In order to validate the screen,the inventors therefore tested chlorpromazine and quinacrine in theiryeast system. As shown in FIG. 7, these two molecules have a certainactivity against the [PSI+] prion. It must however be noted that theiractivities are clearly weaker than that of 6-aminophenanthridine. It canalso be seen that chlorpromazine and quinacrine, like all of themolecules highlighted by the invention, exhibit a strong synergy ofaction with guanidium chloride (In FIG. 7, the medium used contains 200μM of guanidium chloride) The latter result suggests that these twomolecules act on the same biochemical route as the isolated moleculesaccording to the invention.

Moreover, it is interesting to note that quinacrine, the activity ofwhich is approximately ten times greater than that of chlopromazine inProf. Prusiner's test, also exhibits an activity greater than the latterin the screen developed by the inventors. Moreover, just as in Prof.Prusiner's test, chlorpromazine and quinacrine require prolongedtreatment (at least 6 days in the case of Prof. Prusiner's test, atleast two to three days in the case of the screen according to theinvention) before an activity is detected.

Moreover, the inventors determined the activity, in the test accordingto the invention, of other molecules isolated using the test based onmouse neuroblastomas, developed by Prof. Prusiner. A good correlationwas found between the results obtained in the two systems: acepromazinewhich is shown to be slightly active in the mammal system also exhibitsa weak activity in the test according to the invention and the moleculesinactive in analysis on mammals such as carbamazepine, imipramine,haloperidol, chloroprothixene or methylene blue were also inactive inthe test.

Quinacrine has also been described as an inhibitor of multiple drugresistance (MDR). In order to test whether its anti-prion effect couldinvolve this mechanism (which is compatible with the synergic effect ofGuHCl), we evaluated the putative curative effect of an effectivegeneral inhibitor of MDR, verapamil. As shown by FIG. 7, although astrong concentration of this medicament was used, a concentration closeto toxicity, no curative effect could be detected.

All these correlations between the activity of quinacrine andchlorpromazine according to the test or the screen used make it possibleto validate the use of the method according to the invention in order tocarry out high-throughput screenings with a view to isolating moleculescapable of constituting effective medicaments (on mammals and inparticular humans) against neurodegenerative diseases involving proteinaggregates, of spongiform-encephalopathy type, Alzheimer's disease,Huntington's disease etc.

EXAMPLE 8 Analysis of the Inhibition of PrP^(sc) in ScN2a-22L Cells

Mouse neuroblastoma cells infected with the scrapie prion (ScN2a-22L)were used. The cells were cultured in 25 cm² flasks in the presence orabsence of the compounds for several days. Then, the proteins wereextracted from the ScN2a-22L cells by cell lysis in 500 μl of lysisbuffer (50 mM of Tris HCl pH 7.5; 150 mM of NaCl, 0.5% sodiumdeoxycholate; 0.5% Triton X100). After normalization of the proteinswith the Uptima Interchim kit, the adjusted quantities of cell lysateswere digested by proteinase K at 20 μg/ml (Eurobio) for 40 minutes at37° C. The lysates were then centrifuged for 90 minutes at 20,000×g andthe pellet was resuspended in 25 μl of denaturing buffer (1 XTris-Glycine; 4% of SDS, 2% of β-mercaptoethanol; 5% of sucrose andbromophenol blue) and heated for 5 minutes at 100° C. before Westernblot analysis according to the standard protocol using the mousemonoclonal antibody anti-PrP SAF83 (supplied by SPI-BIO,Massy-Palaiseau, France). The percentages of inhibition of the formationde PrP^(sc) resistant to proteinase K were calculated using NIH Image J:the inhibition of the accumulation of PrP^(sc) was 96% forchlorpromazine (Chlor. ) and 70%+/−6% for KP1.

Two of the compounds selected (KP1 and 6AP) were tested in this mammalsystem. As shown by FIG. 8, KP1 was capable of inducing a significantreduction in the accumulation of mammal prion at a dose similar to thatused for chlorpromazine (5 μM). After 7 days of treatment, 70% of thePrP^(sc) resistant to proteinase K have disappeared (wells 1 to 3)compared with untreated cells (wells 4 and 5). This significant effectwas probably under-estimated since the cells treated with the solvent ofthe compounds alone (DMSO 0.01%) showed a significant and reproduciblerise in PrP^(sc) resistant to proteinase K (well 6). The same effect onthe elimination of PrP^(sc) was obtained with 6AP at 2 and 4 μM.

These results therefore validate the use of the screening test accordingto the invention based on yeast in order to isolate anti-prion compoundssince quinacrine and chlorpromazine were detected using this analysisand KP1 and 6AP were also effective in promoting the elimination of themammal prion in vitro.

EXAMPLE 9 Study Structure/Activity

For the purpose of studying the different substitution positions of theanti-prion molecules isolated, the inventors carried out astructure/activity study on the 6-aminophenanthridine molecule.2-fluoro-6-aminophenanthridine (2F-6AP),2-fluoro-6-amino-8-chlorophenanthridine (2f-6A-8ClP) and6-amino-7-chlorophenanthridine (6A-7ClP) molecules were thus obtained bychemical synthesis and their anti-prion activity was determined usingthe test according to the invention. The results obtained are shown inFIG. 9. The diameters of the red halos obtained being proportional tothe anti-prion activity of the molecules deposited, the results indicatethat the presence of a halogen-type substituent at the level ofpositions 7 or 8 increases the anti-prion activity of the molecules offormulae (II) whereas the same type of substituent in position 2 tendsto reduce it.

Bibliographical References

-   Fernandez-Bellot et al., “The protein-only theory and the yeast    Saccharomyces cerevisiae: the prions and the propagons”, CMLS, 2001,    58: 1857-1878.-   Korth C. et al., “Acridine and phenothiazine derivatives as    pharmacotherapeutics for prion disease”, PNAS, 2001, 98(17):    9836-9841.-   Mettey Y. et al., “Synthesis of 11-Aminodibenzo[b,f][1,4]thiazepines    and Fluoro derivatives”, J. Heterocyclic Chem., 1997, 34: 465-467.-   Kessar S. V. et al., Tetrahedron Letters, 1969, 1151.

The invention claimed is:
 1. A pharmaceutical composition comprising: atherapeutically effective quantity of at least one compound of formula(II)

wherein R′ represents an NH₂, or NH—CH(CH₃)—(CH₂)₃—N(CH₂—CH₃)₂ group, Xrepresents F, Cl, or CF₃, p and n, identical or different, are equal to0, 1 or 2, in combination with at least one pharmaceutically acceptablevehicle.
 2. The pharmaceutical composition of claim 1 wherein in thecompound of formula (II), R′ represents an NH₂ group, X represents F,Cl, or CF₃, p and n, identical or different, are equal to 0, 1 or
 2. 3.A pharmaceutical composition comprising: a therapeutically effectivequantity of at least one compound of formula (II)

wherein R′ is —NH—(CH₂)₃—N(CH₃)₂, X is F, Cl, or CF₃, and p and n,identical or different, are equal to 1 or 2, in combination with atleast one pharmaceutically acceptable vehicle.